Genome Mining and Genetic Manipulation Reveal New Isofuranonaphthoquinones in Nocardia Species

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from Nocardia sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of Nocardia sp. CS682 revealed the presence of a furanonaphthoquinone (fnq) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the ifnq gene cluster from Streptomyces sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the Streptomyces antibiotic regulatory protein (SARP) in Nocardia sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called Nocardia.


Introduction
Natural products play an important role in drug discovery, especially in the treatment of various types of cancers and infectious diseases [1][2][3].Quinones are a major class of compounds in the fight against cancers because several of their derivatives have been used as anticancer agents [4,5].However, some toxicity indicators have reduced the pace of global drug development programs.In this regard, new fnqs may serve as lead compounds for the development of anticancer drugs.These compounds are a group of natural compounds distinguished by their unique tricyclic naphtha [2,3-c] furan ring structure, which can have different substituents on rings A, B, or C; these compounds are mostly identified in plants, fungi, and actinobacteria such as Streptomyces sp.CB01883.Ifnqs have shown cytotoxic, antiplasmodial, antioxidant, antibacterial, and siderophore properties [6][7][8][9].In recent years, several specialized metabolites have been discovered through genome mining, which links genetic information with secondary metabolites produced [10].By analyzing the genome databases of diverse actinobacteria, researchers have identified a surprising number of "silent" or "cryptic" biosynthetic gene clusters (BGCs).These clusters are good sources for the discovery of novel and previously unknown metabolites [11].There are several ways to activate such silent BGC to extract new metabolites, such as RNA polymerase mutation, elicitor application, heterologous expression, and overexpression of global or pathway-specific regulators [12,13].
Silent BGCs can be induced to produce potentially novel compounds by altering transcriptional regulators in proximity to BGCs.For example, the overexpression of positive regulators and the inactivation of negative regulators [14] and/or the tuning of the expression of regulators using bldA tRNA [15] have been utilized in various actinomycetes.In the microbial community, Streptomyces antibiotic regulatory protein (SARP) regulators serve as pathway-specific activators of secondary metabolite production [16].In particular, the N-termini of SARP activators have a winged helix-turning helix motif, which is shared by the OmpR protein family.Furthermore, heptameric repeats found in the promoter regions of genes regulated by SARPs can be recognized by at least some parts of SARPs [16,17].Overexpression of the SARP regulator resulted in the activation of some cryptic BGCs and/or enhanced the production of various secondary metabolites [16,18].Recent advancements in different omics techniques, along with the availability of robust genetic engineering techniques such as CRISPR-Cas9, have further facilitated genome mining.
Nocardia sp.CS682 is a well-known producer of nargenicin (ngn) A1, which exhibits potent antibacterial activity against a variety of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA).To produce new secondary metabolites, we engineered a strain, Nocardia sp.CS682DR, by deleting its entire ngn BGC.This led to the characterization of a novel tetrahydroxynaphthalene (THN) derivative, IBR1-4 [19].Bioinformatics analysis revealed the presence of a SARP regulator between the ngn BGC and an unidentified type II PKS.This study aimed to identify and characterize new fnqs with potential therapeutic applications, particularly focusing on their anticancer and antibacterial properties and reducing toxicity.To achieve this goal, we overexpressed the SARP regulator in Nocardia sp.CS682DR, resulting in the production of new ifnq, 6-methoxy-7,8dihydroxy-1-methylnaphtho [2,3-c] furan-9-one (NOC-IBR1) and 6,7-dimethoxy-8-hydroxy-1-methylnaphtho [2,3-c] furan-9-one (NOC-IBR2).Furthermore, we performed gene inactivation using CRISPR-Cas9 to confirm the involvement of the regulator in the biosynthesis of NOC-IBR1/2.Additionally, evaluation of their bioactivities revealed that NOC-IBR2 exhibited significant anti-staphylococcal, anticancer, and antioxidant activities.Remarkably, it was shown that the type II PKS pathway interacts with ThnM3, a methyltransferase located inside the THN BCG.Through this connection, NOC-IBR1 can more easily undergo terminal methylation to yield NOC-IBR2.

Genome Mining
The genome sequence of Nocardia sp.CS682 was analyzed with antiSMASH 7.0 to obtain 46 putative BGCs encoding butyrolactones, ectoine, indole, lantibiotics, melanin, nonribosomal peptides, polyketides, siderophores, terpenes, etc. (Figure S1) [20].The genomic region spanning from 770,703 to 906,587 nt was annotated with type II and type I PKSs, whereas the sequence corresponding to type I PKSs matched the ngn BGC.Bioinformatics analysis of the fnq gene cluster revealed that the SARP regulator gene fnqO is located between two polyketide BGCs via a type I PKS BGC corresponding to ngn and an unknown type II PKS BGC.An in-depth analysis of type II PKSs revealed that they matched the putative BGC for the fnq gene cluster (Table S1).Therefore, the genomic region corresponding to 800,427-826,419 nt was predicted to contain the genes responsible for the biosynthesis of ifnq in Nocardia sp.CS682.

Functional Characterization of the SARP Regulator
The association of transcriptional regulators with secondary metabolites is a wellestablished phenomenon.Overall, SARP regulators are commonly associated with secondary metabolite biosynthesis; hence, we used conventional gene overexpression, deletion, and complementation experiments for the functional characterization of a regulatory gene, fnqO.The production of NOC-IBR1 and NOC-IBR2 in different strains of Nocardia is summarized in Figure 1 and Table S2.In our attempt to evaluate the influence of the SARP regulator (fnqO) bordering the ngn BGC, recombinant pNV18L2-fnqO was introduced into Nocardia sp.CS682 and Nocardia sp.CS682DR to generate Nocardia sp.CS682/fnqO and Nocardia sp.CS682DR/fnqO, respectively.After fermentation, the ethyl acetate extracts of the wild-type and recombinant strains were evaluated by HPLC.The overexpression of fnqO in Nocardia sp.CS682 led to a reduction in nargenicin production at 265 nm according to HPLC (Figure 1b).However, the overexpression of fnqO in Nocardia sp.CS682DR led to low production of NOC-IBR1 (0.8 mg/L) and substantial production of NOC-IBR2 (2.8 mg/L) (Figure 1d).To confirm the close association of fnqO in this regard, we performed a fnqO inactivation experiment using CRISPR-Cas9.The deletion mutant strain was confirmed by PCR amplification and restriction mapping of the amplified fragments (Figure S2).The resulting Nocardia sp.CS682DR∆fnqO completely abolished NOC-IBR1 and NOC-IBR2 (Figure 1e).Additionally, the complementation strain Nocardia sp.CS682DR/∆fnqO/pNV-SARP restored NOC-IBR2 production (Figure 1f); presumably, NOC-IBR1 was converted to NOC-IBR2.Therefore, fnqO is a pathway-specific regulator associated with ifnq production in Nocardia species.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 14 matched the putative BGC for the fnq gene cluster (Table S1).Therefore, the genomic region corresponding to 800,427-826,419 nt was predicted to contain the genes responsible for the biosynthesis of ifnq in Nocardia sp.CS682.

Functional Characterization of the SARP Regulator
The association of transcriptional regulators with secondary metabolites is a wellestablished phenomenon.Overall, SARP regulators are commonly associated with secondary metabolite biosynthesis; hence, we used conventional gene overexpression, deletion, and complementation experiments for the functional characterization of a regulatory gene, fnqO.The production of NOC-IBR1 and NOC-IBR2 in different strains of Nocardia is summarized in Figure 1 and Table S2.In our attempt to evaluate the influence of the SARP regulator (fnqO) bordering the ngn BGC, recombinant pNV18L2-fnqO was introduced into Nocardia sp.CS682 and Nocardia sp.CS682DR to generate Nocardia sp.CS682/fnqO and Nocardia sp.CS682DR/fnqO, respectively.After fermentation, the ethyl acetate extracts of the wild-type and recombinant strains were evaluated by HPLC.The overexpression of fnqO in Nocardia sp.CS682 led to a reduction in nargenicin production at 265 nm according to HPLC (Figure 1b).However, the overexpression of fnqO in Nocardia sp.CS682DR led to low production of NOC-IBR1 (0.8 mg/L) and substantial production of NOC-IBR2 (2.8 mg/L) (Figure 1d).To confirm the close association of fnqO in this regard, we performed a fnqO inactivation experiment using CRISPR-Cas9.The deletion mutant strain was confirmed by PCR amplification and restriction mapping of the amplified fragments (Figure S2).The resulting Nocardia sp.CS682DRΔfnqO completely abolished NOC-IBR1 and NOC-IBR2 (Figure 1e).Additionally, the complementation strain Nocardia sp.CS682DR/ΔfnqO/pNV-SARP restored NOC-IBR2 production (Figure 1f); presumably, NOC-IBR1 was converted to NOC-IBR2.Therefore, fnqO is a pathway-specific regulator associated with ifnq production in Nocardia species.

Identification of the Furanonaphthoquinone Gene Cluster
Bioinformatics analysis [21] of the Nocardia sp.CS682 genome identified the fnq BGC, which showed a high degree of nucleotide sequence identity and a similar genetic organization as the previously reported ifn gene cluster [6].It contains a single type II PKS, which is responsible for the biosynthesis of NOC-IBR1 and NOC-IBR2 (Figure 2A).Analogous proteins from both groups were compared, and the results revealed shared homologs, as shown in Table S1.This finding provides strong evidence that the fnq gene cluster is responsible for the biosynthesis of IFQs, including NOC-IBR1 and NOC-IBR2, in Nocardia sp.CS682.Therefore, we proposed a putative pathway for fnq biosynthesis that utilizes eight molecules of malonyl-CoA processed by Type II PKS and cyclase, resulting in intermediates I and II.Baeyer-Villiger monooxygenases (FnqI) modify polyketide scaffolds (II) synthesized by type II PKS, leading to the creation of product III through a C-C bond cleavage reaction [22,23].Additionally, various putative enzymes participate in the modification of intermediates III to VIII.Intermediate VIII undergoes further modification into NOC-IBR1 and NOC-IBR2 with the involvement of a methyltransferase (ThnM3) (Figure 2B).However, the FNQ BGC lacked an O-methyltransferase (O-MT) responsible for the terminal methylation of NOC-IBR1 to generate NOC-IBR2.Hence, we screened several O-MTs, such as NarM, ThnM1, ThnM2, and ThnM3, for this purpose.Finally, we observed that only ThnM3 was capable of regiospecific methylation [24] to generate NOC-IBR2, as shown in Figure 3.In a previous study, similar metabolites JBIR-76/77 were isolated from Streptomyces sp.RI-77, but their BGCs and pathways were not explored [25].
Bioinformatics analysis [21] of the Nocardia sp.CS682 genome identified the fnq BGC, which showed a high degree of nucleotide sequence identity and a similar genetic organization as the previously reported ifn gene cluster [6].It contains a single type II PKS, which is responsible for the biosynthesis of NOC-IBR1 and NOC-IBR2 (Figure 2A).Analogous proteins from both groups were compared, and the results revealed shared homologs, as shown in Table S1.This finding provides strong evidence that the fnq gene cluster is responsible for the biosynthesis of IFQs, including NOC-IBR1 and NOC-IBR2, in Nocardia sp.CS682.Therefore, we proposed a putative pathway for fnq biosynthesis that utilizes eight molecules of malonyl-CoA processed by Type II PKS and cyclase, resulting in intermediates I and II.Baeyer-Villiger monooxygenases (FnqI) modify polyketide scaffolds (II) synthesized by type II PKS, leading to the creation of product III through a C-C bond cleavage reaction [22,23].Additionally, various putative enzymes participate in the modification of intermediates III to VIII.Intermediate VIII undergoes further modification into NOC-IBR1 and NOC-IBR2 with the involvement of a methyltransferase (ThnM3) (Figure 2B).However, the FNQ BGC lacked an O-methyltransferase (O-MT) responsible for the terminal methylation of NOC-IBR1 to generate NOC-IBR2.Hence, we screened several O-MTs, such as NarM, ThnM1, ThnM2, and ThnM3, for this purpose.Finally, we observed that only ThnM3 was capable of regiospecific methylation [24] to generate NOC-IBR2, as shown in Figure 3.In a previous study, similar metabolites JBIR-76/77 were isolated from Streptomyces sp.RI-77, but their BGCs and pathways were not explored [25].

Evaluation of Microbial Inhibition
The antimicrobial activity of NOC-IBR1 and NOC-IBR2 was evaluated using the paper disk diffusion method.NOC-IBR1 exhibited no antimicrobial activity against the tested pathogens.Conversely, NOC-IBR2 showed significant antimicrobial activity against S. aureus subsp., as evidenced by the clear inhibition zones (Table S3).
As summarized in  NOC-IBR1 and NOC-IBR2 were further subjected to cytotoxicity assays against four different cancer cell lines using a colorimetric test of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Figure 4).The 50% inhibitory concentration (IC 50 ) values of NOC-IBR1 and NOC-IBR2 for the A549, Huh7, HeLa, and U87MG cell lines were 199.5, 44.44, 61.14, 119.6, and 112.5, 54.18, 29.27, and 118.5, respectively, but for the normal HaCaT cell line, the IC 50 value was >200 µM for both compounds.Similarly, the IC 50 values of doxorubicin in the above cell lines were also calculated (Table S4).NOC-IBR2 effectively inhibited the viability of A549 lung cancer cells and HeLa cells compared with NOC-IBR1.These findings further indicate that NOC-IBR1 and NOC-IBR2 substantially reduce the viability of A549, Huh7, HeLa, and U87MG cells in a dose-dependent manner.This is the first report on the cytotoxicity of these two compounds against cancer cell lines.At the concentration of 250 µM, ascorbic acid (positive control) and NOC-IBR2 showed similar ABTS radical scavenging activities (Figure S5).It was observed that NOC-IBR2 showed greater antioxidant activity than NOC-IBR1.

Evaluation of Anticancer and Antioxidant Properties
NOC-IBR1 and NOC-IBR2 were further subjected to cytotoxicity assays against four different cancer cell lines using a colorimetric test of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Figure 4).The 50% inhibitory concentration (IC50) values of NOC-IBR1 and NOC-IBR2 for the A549, Huh7, HeLa, and U87MG cell lines were 199.5, 44.44, 61.14, 119.6, and 112.5, 54.18, 29.27, and 118.5, respectively, but for the normal HaCaT cell line, the IC50 value was >200 µM for both compounds.Similarly, the IC50 values of doxorubicin in the above cell lines were also calculated (Table S4).NOC-IBR2 effectively inhibited the viability of A549 lung cancer cells and HeLa cells compared with NOC-IBR1.These findings further indicate that NOC-IBR1 and NOC-IBR2 substantially reduce the viability of A549, Huh7, HeLa, and U87MG cells in a dose-dependent manner.This is the first report on the cytotoxicity of these two compounds against cancer cell lines.At the concentration of 250 µM, ascorbic acid (positive control) and NOC-IBR2 showed similar ABTS radical scavenging activities (Figure S5).It was observed that NOC-IBR2 showed greater antioxidant activity than NOC-IBR1.

Kinetic Behavior of ThnM3 in Response to NOC-IBR1
The steady-state kinetic parameters of ThnM3 were determined with NOC-IBR1.After determining the impact of the enzyme concentration and conducting a time-course analysis, the initial velocity was measured after 15 min using 5 µg of ThnM3 (O-MT).The estimated apparent K m value of NOC-IBR1 with saturating SAM (2 mM) was 15.62 ± 1.75 µM.The K m value for SAM was estimated to be 39.7 ± 6.08 µM with near-saturating NOC-IBR1 (100 µM) (Figure 5).Table S5 displays the apparent K m and V max values.

Kinetic Behavior of ThnM3 in Response to NOC-IBR1
The steady-state kinetic parameters of ThnM3 were determined with NOC-IBR1.Af ter determining the impact of the enzyme concentration and conducting a time-cours analysis, the initial velocity was measured after 15 min using 5 µg of ThnM3 (O-MT).Th estimated apparent Km value of NOC-IBR1 with saturating SAM (2 mM) was 15.62 ± 1.7 µM.The Km value for SAM was estimated to be 39.7 ± 6.08 µM with near-saturating NOC IBR1 (100 µM) (Figure 5).Table S5 displays the apparent Km and Vmax values.

Microorganisms and Chemicals
The vector used for cloning was pGEM-T Easy (Promega, Madison, WI, USA).Wa tertown, Massachusetts, USA-based Addgene provided the pCRISPomyces-2 vector use in this study.Using the Nocardia-E.coli shuttle vector pNV18, Nocardia sp.CS682 was ge netically modified.DNA manipulation was performed using E. coli XL1 Blue MRF (Strat agene, La Jolla, CA, USA).E. coli ET12567 was used to propagate nonmethylated DNA Every E. coli strain was grown at 37 °C in solid or broth LB media (MB cell, SeoCho-Gu Seoul, Republic of Korea).Brain heart infusion (BHI) (MB cell, SeoCho-Gu, Seoul, Repub lic of Korea) served as the initial medium for seeding and regenerating recombinants, an the DD medium was used as the production medium.The following concentrations o antibiotics were used: 100 µg/mL ampicillin, 50 µg/mL apramycin, and 150 µg/mL thio strepton.Genomic DNA (gDNA) was extracted from Nocardia sp.CS682 using the Qiage DNeasy Tissue Kit (Qiagen, Gaithersburg, MD, USA).gDNA was then used as a templat for PCR amplification.Mallinckrodt Baker (Phillipsburg, NJ, USA) provided acetonitril and trifluoroacetic acid (TFA) of HPLC grade.Additional chemicals were obtained from commercially available sources and were of superior quality.Daejung Chemicals and Met als Co., Ltd.(Shiheung, Republic of Korea) supplied all the remaining organic solvents Sigma-Aldrich (St. Louis, MO, USA) provided dimethyl sulfoxide-d6 (DMSO-d6).Standar molecular biology procedures were followed to isolate, digest, ligate, and manipulat DNA.The recombinant plasmids were verified by DNA sequencing (Macrogen, Inc Seoul, Republic of Korea), and Tables S6 and S7 list all the strains, plasmids, and PCR primers used in this study.

Microorganisms and Chemicals
The vector used for cloning was pGEM-T Easy (Promega, Madison, WI, USA).Watertown, Massachusetts, USA-based Addgene provided the pCRISPomyces-2 vector used in this study.Using the Nocardia-E.coli shuttle vector pNV18, Nocardia sp.CS682 was genetically modified.DNA manipulation was performed using E. coli XL1 Blue MRF (Stratagene, La Jolla, CA, USA).E. coli ET12567 was used to propagate nonmethylated DNA.Every E. coli strain was grown at 37 • C in solid or broth LB media (MB cell, SeoCho-Gu, Seoul, Republic of Korea).Brain heart infusion (BHI) (MB cell, SeoCho-Gu, Seoul, Republic of Korea) served as the initial medium for seeding and regenerating recombinants, and the DD medium was used as the production medium.The following concentrations of antibiotics were used: 100 µg/mL ampicillin, 50 µg/mL apramycin, and 150 µg/mL thiostrepton.Genomic DNA (gDNA) was extracted from Nocardia sp.CS682 using the Qiagen DNeasy Tissue Kit (Qiagen, Gaithersburg, MD, USA).gDNA was then used as a template for PCR amplification.Mallinckrodt Baker (Phillipsburg, NJ, USA) provided acetonitrile and trifluoroacetic acid (TFA) of HPLC grade.Additional chemicals were obtained from commercially available sources and were of superior quality.Daejung Chemicals and Metals Co., Ltd.(Shiheung, Republic of Korea) supplied all the remaining organic solvents.Sigma-Aldrich (St. Louis, MO, USA) provided dimethyl sulfoxide-d 6 (DMSO-d 6 ).Standard molecular biology procedures were followed to isolate, digest, ligate, and manipulate DNA.The recombinant plasmids were verified by DNA sequencing (Macrogen, Inc., Seoul, Republic of Korea), and Tables S6 and S7 list all the strains, plasmids, and PCR primers used in this study.

Genome Mining
The genomic sequence of Nocardia sp.CS682 (GenBank accession no: CP029710.1)was analyzed using antiSMASH 7.0 to annotate 46 putative BGCs (Figure S1) [20].The genomic regions spanning 770, 703-906, and 587 nt containing type II PKSs and type I PKSs were annotated extensively; the sequence corresponding to type I PKSs matched the ngn BGC, and the sequence of type II PKSs matched the putative fnq BGC (Table S1).However, this fnq BGC lacks an O-methyltransferase (OMT) essential for terminal methylation of NOC-IBR1 to generate NOC-IBR2.Therefore, to support this biotransformation, we attempted to identify a suitable OMT among NarM, ThnM1, ThnM2, and ThnM3.

Genetic Manipulation of the SARP Regulator
The presence of a unique SARP regulator adjacent to the ngn BGC spurred our interest in exploring its role in metabolite biosynthesis.For the overexpression of this SARP regulator (FnqO), a codon-optimized gene devoid of the TTA codon (corresponding to the bldA tRNA) was synthesized according to the sequence provided in Table S7 and cloned and inserted into the BamHI and KpnI sites of pNV18L2 to generate pNV18L2-fnqO.This recombinant strain was then transformed into Nocardia sp.CS682 and Nocardia sp.CS682DR through electroporation according to a previously published protocol.The selected transformants were grown at 37 • C for 7 days at 200 rpm (SI 600R Richmond Scientific, Chorley, UK), and secondary metabolites were examined via HPLC.To evaluate the impact of the SARP regulator on secondary metabolite production, we generated a SARP regulator deletion mutant in Nocardia sp.CS682DR using CRISPR-Cas9.For this purpose, pCRISPomyces-2 was used to clone guide RNA (gRNA) and homologous recombination fragments.The gRNA was synthesized as described in Table S7 and ligated to the BbsI site of pCRISPomyces-2 using a previously described protocol [26,27].In contrast, homologous fragments were amplified by PCR with specific primers (Table S7) and cloned and inserted into the XbaI site of pCRISPomyces-2 [27].After the deletion construct was transformed into Nocardia sp.CS682DR, the Nocardia sp.CS682DR∆fnqO strain was successfully generated.After transformation confirmation, the recombinants were cultured on BHI agar plates supplemented with apramycin for additional generations.Then, colonies that did not grow on BHI agar/apramycin plates were selected, and the homologous recombination strain was identified by double crossover.The legitimate deletion strain Nocardia sp.CS682DR∆fnqO was confirmed by PCR and DNA sequencing.The deletion strain was complemented with pNV18L2-fnqO to generate Nocardia sp.CS682DR∆fnqO/pNV18L2-fnqO to evaluate the possibility of polar effects by gene deletion.Metabolites of Nocardia sp.CS682DR and its overexpression, deletion, and complementation strains were evaluated using HPLC and LC-HRMS analyses.

In Vitro Assays of O-Methyltransferase
Following expression, purification, and quantification, ThnM3 was used in the enzymatic reaction to validate its catalytic function.The ability of ThnM3 to introduce a methyl group to NOC-IBR1 was assessed in a general reaction that contained 50 µg of ThnM3, 2 mM SAM, 200 µM substrate, and Tris buffer (50 mM, pH 8) in a final volume of 200 µL.Similarly, a control reaction was performed without heat-inactivated ThnM3.The reaction mixtures were incubated at 40 • C for 30 min.To determine the kinetic parameters of NOC-IBR1, reaction mixtures containing ThnM3 (5 µg), SAM (2 mM), Tris buffer (50 mM, pH 8), and various concentrations of NOC-IBR1 (5-200 µM) were prepared in a total volume of 200 µL.Similarly, 200 µL reaction mixtures comprising ThnM3 (5 µg), NOC-IBR1 (100 µM), Tris buffer (50 mM, pH 8), and different concentrations of SAM (5-200 mM) were prepared to determine the kinetic parameters of ThnM3 for SAM.Different enzyme concentrations for ThnM3 assays were tested for 30 min in a 200 µL reaction mixture containing ThnM3 (1-5 µg), 5 µM NOC-IBR1, 2 mM SAM, and Tris buffer (50 mM, pH 8).ThnM3 (5 µg), 2 mM SAM, 5 µM NOC-IBR1, and Tris buffer (50 mM, pH 8.0) were used to measure the time-dependent in vitro conversion of NOC-IBR1 to NOC-IBR2 every 5 min for 30 min.To stop each reaction, 200 µL (double volume) of chilled methanol was added, and HPLC was used to examine the supernatant after 30 min of centrifugation at 10,000× g.Data were obtained in triplicate, and appropriate control reactions were maintained.The data are reported as the mean and standard deviation.Nonlinear regression analysis was used to calculate the values of Vmax and the Michaelis-Menten constant, K m , under the assumption of steady-state Michaelis-Menten kinetics [28].

Fermentation and Extraction
Five hundred-milliliter flasks containing 50 mL of BHI medium were used to cultivate Nocardia sp.CS682DR as well as the overexpression, deletion, and complementation strains.Following a two-day incubation at 37 • C, 20 mL of seed culture was added to each of the 5 L fermenters (XP-50 Fermentec, Republic of Korea), which contained 3 L of DD medium (0.2% oatmeal, 0.2% yeast extract, 0.2% soybean meal, 0.1% calcium chloride, 1% maltose, 0.1% magnesium chloride, and 0.4% glycerol) for fermentation in a bioreactor.The fermenters were shaken at 400 rpm and 37 • C.After 5 days, the resulting culture was mixed with a double volume of ethyl acetate and shaken for 5-7 h.A rotary evaporator was used to dry the extracts, and methanol was added.The concentrated sample was filtered with an HPLC Syringe Filter (Catalog; 10463060, Whatman, UK).Subsequently, 20 µL of the filtered extracts were analyzed using reverse-phase HPLC-PDA coupled with a C18 column (Mightysil RP-18 GP (4.6 × 250 mm, 5 µM) at 320 nm) to identify peaks corresponding to various secondary metabolites, including NOC-IBR1/2.Binary conditions were used with solvent 1 (water containing 0.05% trifluoroacetic acid buffer) and solvent 2 (100% acetonitrile (ACN)), maintaining a flow rate of 1 mL/min for 30 min; gradient conditions included 10% (0-2 min), 70% (20-24 min), 100% (24-28 min), and 50% (28-30 min) ACN.HR-qTOF ESI/MS analysis was performed with SYNAPT G2-S (Waters Corp., Milford, MA, USA) in a positive ion mode.The target compounds (NOC-IBR1/2) were purified by prep-HPLC using a C18 column (YMC-Pack ODS-AQ-HG, 250 × 20 mm, 10 µM, Shimogyo-Ku, Kyoto, Japan) coupled to a UV detector (320 nm) and a 40 min binary gradient at a flow rate of 10 mL/min with 1.2 mL injection volume to the loop volume of 2 mL.The sample was filtered with an HPLC Syringe Filter (Catalog; 10463060, Whatman, UK).The concentration of ACN in the binary gradient was as follows: 0-10 min, 0-15%; 15-20 min, 50%; 20-25 min, 80%; 25-24 min, 95%; 34-39 min, 10%; and finally stopped at 40 min with a flow rate of 10%.The peaks for NOC-IBR1 and NOC-IBR2 corresponding to retention times (t R ) of 26 min and 29 min in prep-HPLC with a minimal UV threshold of 1000 U, respectively, were collected.The collected fractions from prep-HPLC were reanalyzed using analytical HPLC to determine their purity.Following lyophilization, the purified products were dissolved in deuterated DMSO-d 6 obtained from Sigma-Aldrich and subjected to nuclear magnetic resonance (NMR) spectroscopy (700 MHz) to determine their structures.For structural confirmation, two-dimensional nuclear magnetic resonance (NMR) techniques such as heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) were used in addition to 1 H-NMR and 13 C-NMR.

Antimicrobial Assays
Antimicrobial assays of NOC-IBR1 and NOC-IBR2 were carried out against a panel of eighteen different pathogens.Among them, fourteen were Gram-positive bacteria ).The paper disk diffusion assays on Mueller-Hinton agar plates (MHA) were performed according to the Kirby-Bauer method and Clinical Laboratory Standard Institute (CLSI) guidelines [29,30].NOC-IBR1 and NOC-IBR2 were applied to paper disks to adjust the final concentration to 40 µg/disk.MHA plates were inoculated with 1 × 10 8 colonyforming units (CFUs)/mL of bacterial culture.Using sterile paper plates measuring 6 mm (Advantec, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), a 40 µg/disk compound was applied to the surface of the infected agar plates.The samples were then incubated for 18-20 h at 37 • C. The inhibition diameter zone was then measured, calculated for each pathogen, and expressed in millimeters (mm).Since all the compounds were dissolved in dimethyl sulfoxide (DMSO), it was used as a solvent control, and erythromycin was used as a positive control.
The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of NOC-IBR1 and NOC-IBR2 were estimated using a previously described method [31].The broth dilution method was then used to calculate the MICs of NOC-IBR1, NOC-IBR2, and erythromycin as positive controls against nine strains of the Gram-positive bacterium S. aureus subsp.Mueller-Hinton broth (MHB, Difco, Baltimore, MD, USA), and the assays were performed in duplicate in 96-well plates.The nine MRSA strains were subsequently grown in MHB and subjected to MIC and MBC assays.To determine the MIC, the broth dilution method was applied [32].In a 96-well plate, MHB and the samples were dispensed and diluted serially.In all the wells except for the negative control (broth only), the bacterial cell suspension was adjusted to 0.5 McFarland standard (1 × 10 8 CFU/mL) and diluted to 2.5 × 10 6 CFU/mL in MHB.The cells were then cultured for 16-20 h at 37 • C. The MBC assay was carried out on an MHB medium by inoculating cultured samples containing MIC compounds for 18 h at 37 • C, and the MICs of the bacterial strains were determined.The assays were repeated twice to maximize reliability and reproducibility.
The ABTS radical cation decolorization assay was performed to evaluate the free radical scavenging potential of NOC-IBR1 and NOC-IBR2 using ascorbic acid as a standard reference [33,34].The decrease in absorbance signifies the ability of the samples to neutralize or scavenge the ABTS + • radical, indicating their free radical scavenging capacity.The ability of NOC-IBR1 and NOC-IBR2 to eliminate free radicals was evaluated using the ABTS radical cation decolorization test [35].ABTS (7 mM) in deionized water and 2.45 mM potassium persulfate (1:1) were combined to form the ABTS + • cation radical, which was then kept at room temperature in the dark for 12 to 16 h before use.After that, ethanol or deionized water was added to the ABTS +• solution to dilute it until the absorbance at 734 nm was 0.70.In 96-well plates, 10 µL of the sample was combined with 195 µL of ABTS +• solution, and in the blank well, 195 µL of ABTS + solution was combined with 10 µL of DMSO and incubated at room temperature for 30 min.Then, the absorbance was measured at 734 nm.Ascorbic acid was used as a standard reference, and all determinations were performed in triplicate.

Statistical Analysis
The findings are presented as the means ± standard errors (SEs).Statistical significance between the two groups, comprising the control and test groups, was assessed using the Student's t-test.A p-value less than 0.05 was considered indicative of statistical significance.

Figure 2 .
Figure 2. (A) Organization of the NOC-IBR1 and NOC-IBR2 biosynthesis-related gene clusters from Nocardia sp.CS682DR; the translational directions of the genes are indicated by arrows, and the letters above the arrows represent the gene names.(B) Proposed biosynthetic pathway for compounds NOC-IBR1 and NOC-IBR2, where FnqE1/E2/E3 are acyl carrier proteins (ACPs), FnqI is a Bayer-Villiger monooxygenase, and FnqA is an esterase.FnqGH = SnoaL-like domain protein and FAD oxidase; FnqJ = hydrolase.FnqBCD = ferredoxin, cytochrome P450, and flavin reductase, respectively; ThnM3 = methyltransferase.fnqO in the orange frame is the SARP gene.

Figure 2 .
Figure 2. (A) Organization of the NOC-IBR1 and NOC-IBR2 biosynthesis-related gene clusters from Nocardia sp.CS682DR; the translational directions of the genes are indicated by arrows, and the letters above the arrows represent the gene names.(B) Proposed biosynthetic pathway for compounds NOC-IBR1 and NOC-IBR2, where FnqE1/E2/E3 are acyl carrier proteins (ACPs), FnqI is a Bayer-Villiger monooxygenase, and FnqA is an esterase.FnqGH = SnoaL-like domain protein and FAD oxidase; FnqJ = hydrolase.FnqBCD = ferredoxin, cytochrome P450, and flavin reductase, respectively; ThnM3 = methyltransferase.fnqO in the orange frame is the SARP gene.

Figure 4 .
Figure 4.The cytotoxicity of NOC-IBR1 and NOC-IBR2 was assessed across different cell lines.Each cell line was maintained in a 5% CO2 humidified incubator at 37 °C.The cells were seeded at a

Figure 4 .
Figure 4.The cytotoxicity of NOC-IBR1 and NOC-IBR2 was assessed across different cell lines.Each cell line was maintained in a 5% CO 2 humidified incubator at 37 • C. The cells were seeded at a density of 2 × 10 3 cells/well in 96-well culture plates for the cell growth assay.Various concentrations of the compounds (200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.16 µM, 1.56 µM, and 0.78 µM) were added to each well, and the cells were incubated for 72 h.Cell growth was measured using the MTT colorimetric assay.

Table 2 .
MIC and MBC of NOC-IBR2 and positive control erythromycin (Erm) against nine strains of MRSA.

Table 2 .
MIC and MBC of NOC-IBR2 and positive control erythromycin (Erm) against nine strains of MRSA.